Figure 2.

Notch activation increases the level of phosphorylated CREB in adult brains. A, Induction of Notch activity triggers the accumulation of phosphorylated CREB protein in the brain in a pattern similar to Notch protein expression. Highly cross-absorbed AlexaFluor-647 (against chicken anti-Notch) and Alexa-488 (against rabbit anti-PO4 CREB) secondary antibodies were used to eliminate cross-reaction and signal bleed between channels. Imaging was done under identical settings. Similar results were obtained with N^nd1 flies. B, Hyper-PO4 CREB is very unstable in adult brains. Any delay in the crushing of detached adult heads with 1× Laemmli buffer results in the loss of hyper-PO4 CREB, concomitant with the accumulation of smaller forms (#). Flies were incubated at 30°C for 30 min, then at room temperature for 10 min, immobilized on ice-chilled Petri plate platform (<30 s), the heads chopped with a blade, transferred to a 0.6 ml microfuge tube with 1× Laemmli buffer (individually or in batches of five), crushed with a pestle, and boiled immediately. The blots were probed with the PO4 CREB antibody. Hyper-PO4 CREB is clearly detectable in one-head-at-a-time N^nd3 sample (lanes 3 and 4) but not in 5-heads-at-a-time N^nd3 sample (lanes 1 and 2). C, The recovery of hyper-PO4 CREB is markedly improved when chilling for fly immobilization is eliminated, and the fly head is detached and crushed within 5 s after capture of the fly with a mouth-operated aspirator. Lane 5 shows CREB isoform levels in the sample where whole flies were instantly crushed in 1× Laemmli (∼2 s to crushing). A comparison of the levels of the hypo-PO4 CREB 1 and 2 in lanes 1–3 with those in lane 5 suggests that stress-related alteration in the levels of CREB isoforms was not eliminated even in the aspirator-based, rapid capture-chop-crush head-protein extraction procedure.